Efficient Building Blocks for Solid-Phase Peptide Synthesis of Spin Labeled Peptides for Electron Paramagnetic Resonance and Dynamic Nuclear Polarization Applications.

Specific spin labeling allows the site-selective investigation of biomolecules by EPR and DNP enhanced NMR spectroscopy. A novel spin labeling strategy for commercially available Fmoc-amino acids is developed. In this approach, the PROXYL spin label is covalently attached to the hydroxyl side chain of three amino acids hydroxyproline (Hyp), serine (Ser) and tyrosine (Tyr) by a simple three-step synthesis route. The obtained PROXYL containing building-blocks are N-terminally protected by the Fmoc-protection group, which makes them applicable for the use in solid-phase peptide synthesis (SPPS). This approach allows the insertion of the spin label at any desired position during SPPS, which makes it more versatile than the widely used post synthetic spin labeling strategies. For the final building-blocks, the radical activity is proven by EPR. DNP enhanced solid-state NMR experiments employing these building-blocks in a TCE solution show enhancement factors of up to 26 for 1 H and 13 C (1 H→13 C cross-polarization). To proof the viability of the presented building-blocks for insertion of the spin label during SPPS the penta-peptide Acetyl-Gly-Ser(PROXYL)-Gly-Gly-Gly was synthesized employing the spin labeled Ser building-block. This peptide could successfully be isolated and the spin label activity proved by EPR and DNP NMR measurements, showing enhancement factors of 12.1±0.1 for 1 H and 13.9±0.5 for 13 C (direct polarization).

Combining Freezing Point Depression and Self-Diffusion Data for Characterizing Aggregation.

The colligative property freezing point depression is evaluated as a means for estimating the extent of aggregation for solutions of poly(ethylene oxide) alcohol (C10E6) nonionic surfactant in cyclohexane. Combined with additional measurements of self-diffusion coefficients, it is shown that both unaggregated C10E6 as well as reverse micelles are significantly present for the entire range of measured C10E6 concentration (0.048-2.35 mol kg-1). A change in speciation near 0.2 mol kg-1 is indicated by the results from both freezing point depression and self-diffusion coefficient measurements. It is shown that average reverse micelle radii and aggregation numbers obtained from the ratio of solvent and C10E6 self-diffusion coefficients are consistent with prior reported results. However, unreasonably small radii for the reverse micelles as well as for the cyclohexane were obtained from analysis of the results by the Stokes-Einstein equation using additional measured solution viscosities. The concentration of reverse micelles and unaggregated C10E6 was calculated from the freezing point depression results using the aggregation numbers obtained from ratio of self-diffusion coefficients. These concentrations indicate that the reverse micelles become smaller in average size and increase in number with increasing temperature without an increase in unaggregated C10E6.